The differentiating erythroid cell, particularly in thalassemia, is exposed to severe cellular stresses including oxidative and hypoxic stress. Both reactive oxygen species and hypoxia lead to the phosphorylation of eIF2a, a translation factor vital for the initiation of protein synthesis, and genetic studies have demonstrated that the regulation of this phosphorylation plays an important role in erythropoiesis. We have determined that the inhibition of nonsense mediated RNA decay in hypoxic cells is dependent on eIF2a phosphorylation. Nonsense mediated RNA decay (NMD) is a multi-step pathway responsible for the degradation of 30% of all human mutated mRNAs, as well as up to 10% of normal cellular mRNAs. But because NMD has not been thought of as a regulated pathway, its activity in normal or thalassemic erythroid cells has not been closely studied, and its significance in erythropoiesis has not been determined. We hypothesize that NMD is inhibited by cellular hypoxia and reactive oxygen species in differentiating thalassemic erythroid cells. This inhibition, mediated by eIF2a phosphorylation, alters gene expression, augments the stress response, and improves the survival of these cells. We propose to 1) Determine if eIF2a is phosphorylated by reactive oxygen species in human thalassemic erythroid cells, and whether this phosphorylation is sufficient for the inhibition of NMD. Briefly, we will amplify and differentiate erythroid cells from peripheral blood, and also examine bone marrow biopsies, from control and thalassemic volunteers. These samples will be assessed for eIF2a phosphorylation status, induction of stress responsive genes, and reactive oxygen species. We will then use a variety of stresses and engineered cell lines to determine if eIF2a phosphorylation is sufficient to inhibit NMD. 2) Determine the biological significance of eIF2a phosphorylation and NMD inhibition in normal and thalassemic erythroid cells. Briefly, we will determine if eIF2a phosphorylation by reactive oxygen species or cellular hypoxia correlates with apoptosis in differentiating erythroid cells. Using expression arrays we will determine the mRNAs that are stabilized when NMD is genetically deleted in erythroid cells and/or when NMD is inhibited by hypoxia in these cells. NMD targets alternatively spliced mRNAs, and by using expression arrays that identify mRNA splice variants we will determine if these variants are enriched in hypoxic and NMD repressed erythroid cells. 3) Determine the mechanism of hypoxia-induced inhibition of nonsense mediated RNA decay by pursuing our working model, based on preliminary data, that eIF2a phosphorylation sequesters NMD targeted mRNAs to cytoplasmic stress granules, where these mRNAs cannot be degraded. Using confocal microscopy we will determine the localization of enzymes important for NMD, as well as NMD degraded mRNAs in stressed cells where eIF2a is phosphorylated. PUBLIC HEALTH RELEVANCE: The normal growth and development of red blood cells is vital to sustain health. Impairment of red cell development, in diseases such as thalassemia, may lead to a severe anemia and a dependence on transfusions. We have identified a novel mechanism that controls the stability of messenger RNA, the carrier of genetic information from DNA to proteins in a cell. Because this mechanism is regulated by cellular stresses found in the normal, and particularly thalassemic, erythroid cells, we will determine if erythroid cell survival is affected by this novel form of gene regulation, and identify the genes that are regulated by this mechanism.